Cloning, sequencing, and functional analysis of a Marek's disease virus origin of DNA replication.

0022-538X/91/116320-05$02.00/0

Cloning, Sequencing,

and Functional

Analysis of

a

Marek's Disease

Virus

Origin of DNA

Replication

HEIDI S. CAMP,1 PAUL M. COUSSENS,1 ANDROBERT F. SILVA2*

Molecular VirologyLaboratory, Department of Animal Science, MichiganState University,EastLansing, Michigan

48824,1

andAvianDisease and

Oncology Laboratory,

AgriculturalResearchService, U.S.Department ofAgriculture,

3606 East MountHope Road, EastLansing, Michigan488232

Received6May 1991/Accepted30July 1991

Previously, we isolated a replicon from a defective Marek's disease virus (MDV), analogous to defective herpes simplex viruses (amplicons). Defective viruses containcis-actingelementsrequiredfor DNAsynthesis

andviruspropagationsuch as anoriginof DNAreplicationand apackaging-cleavage signalsite. In thisreport, the MDVrepliconwas utilized to locate anorigin ofMDVDNAreplication. A comparisonof MDVreplicon sequences with other herpesvirus replication origin sequences revealed a 90-bp sequence containing 72%

identity to thelytic origin

(oris)

ofherpessimplexvirustype1. This90-bp sequence displayednosimilarityto

betaherpesvirus or gammaherpesvirus replication origins. The 90-bp sequenceis arranged as an imperfect palindrome centered around an A+T-rich region. This sequence also contains a 9-bp motif (5'CGTTCG CAC3') highlyconserved in alphaherpesvirus replication origins. To test functionalityof the 90-bpputative MDVreplicationorigin,weconductedDpnI replication assays with subclonesgeneratedfrom the4-kbpMDV

replicon.A700-bpMDVreplicon subfragment containingthe90-bpputativeMDVreplicationorigin sequence

iscapableofreplicatinginchickenembryofibroblast cells cotransfected withhelpervirus DNA. Inconclusion, weidentified afunctionalorigin ofDNAreplicationin MDV. Similarityof MDVorigin sequencestothoseof

alphaherpesviruses supportsthe currentcontentionthat MDV is moreclosely relatedto alphaherpesviruses

than togammaherpesviruses.

Marek's disease virus (MDV) is a highly cell-associated avian herpesvirus. Inchickens, MDV is the etiologic agent ofMarek'sdisease, amalignant T-celllymphoma(7). There are three MDV serotypes. Serotype 1 includes oncogenic MDVsand their attenuated derivatives, serotype 2 includes the closely related, naturally occurring nononcogenic chicken herpesviruses, and serotype 3 is the antigenically related nononcogenic turkey herpesviruses. MDV pathology has beenextensively characterized (7, 23). Molecular anal-ysis of MDV, however, has lagged behind that of other herpesviruses, primarily because of technical difficulties presented by the tightly cell-associated nature of MDV infection.

The MDV genome, a double-stranded linear DNA mole-cule of approximately 160 to 180 kbp, consists of a unique long (UL) and a unique short

(Us)

segment flanked by inverted repeats (TRL, IRL,

IRS,

TRS). MDV has been classified as a gammaherpesvirus based on its lymphotro-pism (25). However, the overall genomic structure and colinearity of many MDV genes to those of alphaherpesvi-ruses such as herpes simplex virus (HSV) and

varicella-zostervirus suggest that MDV should be reclassified (5). Previously, we reported the isolation and characterization of a defective serotype 2 MDV (8). The defective MDV (replicon)genomeexists as a high-molecular-weight head-to-tail concatemer consisting of 4-kbp viral monomeric repeats and appears to be analogousto HSV amplicons (12). A 4-kbp repeat was cloned into the EcoRI site in pUC19 and was designated pA5 (8).

HSV amplicons contain multiple head-to-tail reiterations of monomeric repeat units derived from either end of the

Us

fragment orfrom two noncontiguous regions within the

Us

* Corresponding author.

andULfragmentsof the HSV genome(11). HSV amplicons replicateand propagate in the presenceofahelpervirus(11, 12, 19, 30, 34, 35). Molecular analysis of HSV amplicons revealed three lytic replication origins (oriL andtwocopies

of

oris)

andapackaging-cleavage signalsite within the HSV

genome (11, 12, 30, 31, 33-35).

DNA sequencing. DNA sequencing of the 4-kbp MDV replicon was initiatedtoidentify a potential origin of repli-cation in MDV. pA5Iwas constructed by cloningthe 4-kbp replicon into pUC19in theinverse orientationwith respect to pA5. Unidirectional deletion mutants were created from pA5 andpA5I with exonuclease III and Si nuclease (Boehr-ingerMannheimBiochemicals,Indianapolis, Ind.), and then deleted cloneswere recircularized with T4 DNA ligase (16). DNAsequencingwasperformedwithdouble-stranded plas-mid templates and the dideoxy chain termination method (26). In all sequencing reactions, the Sequenase enzyme (United States Biochemical Corp., Cleveland, Ohio) was usedas recommended bythe manufacturer. MDV replicon sequences were compared with sequences in the GenBank data base with the MacVector (International Biotechnolo-gies, Inc., NewHaven, Conn.) computer program.

A90-bp A+T-rich sequence, closely related to HSV and varicella-zoster virus replication origin sequences, was lo-cated betweenaPstI-to-BdnHI site of pA5 (9, 20, 33) (Fig. 1A). Overall, the 90-bp sequence contains 72% sequence identityto core regions of the HSV-1

oris

andoriL and 66% sequence identity to origins of varicella-zoster virus and equineherpesvirus 1 replication (2, 9, 20, 24, 32, 33) (Fig. 1B). Neither the replication origins of Epstein-Barr virus (oriP and oriLyt) nor those of cytomegalovirus contained sequences similarto theputative origin of serotype 2 MDV replication (1, 13, 14, 15, 37).

The 90-bp sequence is arranged in an imperfect palin-dromecontaining30bp of alternatingATresidues, whereas 6320

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A. NJV ori

10 20 30 40 50

A0XCICAC G(iiGCACCG CGAACCAATA TAPGATTATA TATATAATAT

60 70 80 90

ATIATGCG

CAGG'IGA_ACCCCrC

03CAATICGGG

10 20 30 40 50 60 70 80

B. MDV ori ACGCGTCAGC GTTCGCACCG CGAACCAATA TAAGAITATA TATATAATATAITATTGGCG CAAGGTGCGA ACGCCCGTCC GCGCAATCGG

HSV-1 oris ACGCG-.AGC GITCGCAC.. CG..CCAATA T---A-TATA TATAT----T ATrA- .GGCG -AA-GGTGCGA AC...CG.C. GC.

HSV-1 oriL ACGCG-.AGC GTTCGCAC. G-.CC.A-A TAA--- TATA TATAT ----T ATTA--GG-. CAA.IGTGCGA ACG- -C.TC. G

VZV ori ---.CA.C GTTCGCAC...ATA T-A.A-TATA TATAT-ATAT A-TAT.G.-G -AA.G.G.GA ---- GT

EHV-1 oris -.G.G. -..C GTTCGCACC. ...ACCA- -A TAA-.T-.-. T-.ATAAT-. ATTATT... CAA.GGTGO3GA AC.

FIG. 1. Identification of a putative origin of MDV serotype 2 replication by DNA sequence analysis of the MDV replicon. DNA sequencingwasperformed by the dideoxy chain termination method. (A) Nucleotide sequence of the 90-bp putative origin of MDV serotype 2 replication. Black bars indicate a 9-bp sequence shown to be recognized by the HSV-1 origin-binding protein. (B) Alignment of MDV serotype 2replication origin withalphaherpesviruslytic origins ofreplication. Black bars indicate highly conserved sequences among other origins of herpesvirus replication. Dots (.) represent mismatched sequences, and dashes (-) indicate gaps.

the

oris

and

oriL

of HSV contain a nearly perfect palindrome with an 18-bp A+T-rich region (9, 33). Lockshon and Galloway (20) reported that the palindrome center, contain-ing the 18-bp A+T-rich sequence, is essential for HSV type 2 (HSV-2) replication. However, expansion of the 18-bp A+T-rich region to 52 bp by introducing alternating AT sequences did not abolish the HSV-2 replication function (20). Interestingly, theputative MDV replication origin also contained a 9-bp sequence (CGTTCGCAC) that is highly conservedinalphaherpesvirus replication origins (2, 24, 32, 33). This 9-bp sequence is a subset of an 11-bp motif (CGTTCGCACTT)shown to be recognized by the HSV type 1 (HSV-1) origin-binding protein, the product of UL9 gene expression (10, 18). Recently, Bruckner et al. (4) reported that the HSV-1 origin-binding protein contains helicase activity, possibly used for unwinding DNA duplex at the origin. Othershaverecentlyreportedthat properinteraction of origin-binding protein with

oris,

containing the 11-bp motif, isessential forDNA replication(17).

Plasmid replication. To determine whether the putative MDV origin, identified by DNA sequence analysis, could function as areplication origin, we constructed a series of subclones from pA5 (Fig. 2). pNOTA5 was derived from pA5 byreplacing a2.5-kbp HpaI and ClaIfragment of pA5 withNotIlinkers(agiftfrom A. Finkelstein). p281MI-1 was constructed by cloning a 2-kbp BamHI fragment from the 281MI/1 genome into pUC18 at the BamHI site.

pCK300

containsa300-bp ClaI-to-KpnI

subfragment

of

pA5

inserted between the ClaI and KpnI sites in

pUC18.

A

700-bp

PstI-to-KpnI subfragment of pA5 was isolated and cloned intopUC18 byusingthe PstI andKpnIsitestocreate

pA700.

All pA5 subclones as well as pA5 were

propagated

in Escherichia coli DH5a competent

cells,

a

Dam'

E. coli strain.Also,the4-kbpEcoRI

replicon

fragment

wasisolated frompA5and

religated.

We conducted DpnI resistance assays

(33)

on

pA5

sub-clones as well as the circular form of the

4-kbp

replicon.

DpnI cleaves only

methylated

GATC sequences;

therefore,

DNApropagated from

Dam'

strainsofE. coli is

methylated

at the adenine residues within GATC and

susceptible

to DpnI digestion. DNA

replicated

in

eucaryotic

cells is not methylatedatGATC sequences and is therefore resistantto DpnI cleavage.

High-molecular-weight

DNA was isolated from chicken embryo fibroblast(CEF) cells infectedwith

281MI/1

passage

15, aserotype 2 MDV(36). Preparation andmaintenanceof primary or secondary CEF cultures have been previously described (28). Virus stocks were determined to be free of reticuloendotheliosis and avian leukosis viruses by either complement fixation or enzyme-linked immunosorbent

as-say(29). Upon evidenceof extensivecytopathiceffect,cells

were lysed in 150 mM NaCl-100 mM EDTA-1% sodium dodecyl sulfate-100 ,ugofproteinase Kpermlfor 4 to24h

at 37°C. DNAwasextracted twice withanequalvolume of phenol-chloroform-isoamylalcohol(25:24:1,vol/vol/vol)and

once with chloroform-isoamyl alcohol (24:1,

vol/vol)

and precipitated with 2.5 volumes of100% ethanol. The DNA

wasresuspendedin TE (1mMEDTA, 10 mMTris-HCl, pH 8.0)and stored at4°C.

Secondary CEF cells were cotransfected with 500 ng of

pA5

pA700

E Hpl Sm P RH K Cl H3 Hpl BH P Sm Hl E

/

\I

Ilm

I

P BwiK

I~

pCK300

E HDI p281MI-1

E H I

pNOTAS

BH Psm HIE

Cl H3 Hpl\ S H[E

FIG. 2. Generation of pA5 subclones. All subclones were

in-serted into pUC18. The location of the

putative

MDV origin of

replication is indicated as an open circle (o). E, EcoRI;

Hpl,

HpaI;Sm,SmaI;P,PstI;BH,BamHI;K, KpnI;Cl,ClaI;andH3, HindIll.

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0

ee

55.5

5.2-2

4.0_op

2.8.a

I..

1 2 3 4 5

FIG. 3. Replication of the 4-kbpreplicon and pA5. The 4-kbp

replicon andpA5weresubjectedtotheDpnI resistanceassaytotest their abilitytoreplicate in CEF cells. CEF cellswerecotransfected

with eitherthe circularform of the 4-kbpreplicon DNA(lane 3)or

pA5 (lane 4) and 281MI/1 DNA. Seven days aftercotransfection,

total cellular DNA was digested with EcoRI and DpnI, resolved

througha0.8%agarosegel, and transferredtoanylon membrane.

The membrane was probed with 32P-labeled 4-kbp replicon DNA

andvisualized by autoradiography. Lanes: 1, mock-infected CEF

DNA; 2,DNA from CEFtransfected with281MI/1DNA; 3, DNA

from CEFcotransfectedwith281MI/1DNAand thecircular form of

the 4-kbp replicon DNA; 4, DNA from CEF cotransfected with

281MI/1 DNA and pA5; 5, 281MI/1 DNAmixed with methylated

inputpA5.Numbersonleft show size in kilobasepairs.

plasmid DNA and 20 ,ug of 281MI/1 DNA by calcium phosphate coprecipitation (22), exceptthat Dulbecco

mini-mal essentialmediumwasused throughout theexperiments.

When 80% of CEFmonolayersexhibited cytopathic effect(5

to 8 days after cotransfection), total high-molecular-weight

cellularDNA was isolatedas described above.

Approximately 6,ug oftotal cellular DNA was digested with EcoRI andDpnI (International Biotechnologies, Inc.)

under conditions recommended by the manufacturer.

Di-gestedDNA fragments wereresolvedon 0.8%agarose gels

andtransferredtoZetaProbe membranes (Bio-Rad

Labora-tories, Inc., Richmond, Calif.) as recommended by the

supplier. Probes were prepared with [a-32P]dCTP with a

randomprimed labeling kit (Bethesda Research

Laborato-ries,Inc., Gaithersburg, Md.) as specified by the

manufac-turer.Nylonmembranesweresubsequently hybridized with

appropriate32P-labeled probes(6).

As expected, EcoRI and DpnI cleavage of

281MI/1-in-fected CEFDNAproduced5.5-, 5.2-, and 2.8-kbp fragments

which hybridizedtothe4-kbp repliconprobe (Fig. 3,lane2).

The 4-kbp replicon probe, however, did not

hybridize

to uninfected CEF DNA(Fig. 3, lane1).

Cotransfection

ofCEF cells with circular

EcoRI

4-kbp DNA and

281MI/1

DNA resulted in a

DpnI-resistant

EcoRI

fragmentof4kbp

(Fig.

3, lane 3) in addition to the three

EcoRI

fragments from the

281MI/1

helpervirus genome. DNAisolatedfrom CEF cells cotransfected with intactpA5and

281MI/1

DNA also

yielded

a 4-kbp

EcoRI

fragment resistant to

DpnI

cleavage

(Fig. 3,

lane 4) in addition to the three

EcoRI

fragments.

Methylated

inputpA5 DNA mixed with

281MI/1-infected

CEF DNAwas cleaved by

DpnI,

indicating that

DpnI

resistance was not a result of inhibition of

DpnI

activity (Fig. 3, lane 5).

Results of the

DpnI

resistance assay confirmed that the 4-kbp circular form of the MDV replicon was able to

replicate in CEFcells in the presenceof

281MI/1

helper

virus DNA. pA5, containing the 4-kbp replicon inserted into the

EcoRI

site of pUC19, was also replicated as anepisome in CEF cells cotransfected with

281MI/1

helper virus DNA. Therefore, we concluded that sequences spanningthe

EcoRI

site within MDV replicon DNA are not required for ampli-fication of replicon DNA. However,replication ofthe 4-kbp replicon was at least fivefold more efficient than thatof pA5. Insertion of pUC19 sequences within the

EcoRI

site of replicon DNA may have reduced the ability of pA5 to replicate. The decrease in replication activity of pA5 may also be due to some unknown inhibitory sequences within pUC19.

Replication activity of pA5 subclones was indicated by the presence of unit-length

DpnI-resistant

EcoRI

subclone DNA following digestion of cotransfected total cell DNA with EcoRI and

DpnI

(EcoRI

introduces a single cut within the pA5 subclones). During cotransfection experiments, we ob-served MDV plaque reduction by certain pA5 subclones. Thus, 32P-labeled pA5 was used as a probe to detect both plasmid DNA as well as helper virus DNA. As expected, intact pA5 was able to replicate in the presence of helper virus DNA (Fig. 4, lane 5), generating

DpnI-resistant

EcoRI

fragments of 4 and 2.7 kbp corresponding to virus- and plasmid-specific bands, respectively. However, pA5 alone did not show replication activity, suggesting that functions provided in trans by a helper virus are essential for replica-tion (Fig. 4, lane 3). Methylated input pA5, used as a control, was susceptible to

DpnI

cleavage (Fig. 4, lane 1). Among tested subclones, only pA700, which contains the putative MDV origin of replication identified by DNA sequence analysis, replicated as indicated by a

DpnI-resistant

EcoRI

fragment of 3.4 kbp (2,686-bp pUC18 plus 700-bp

PstI-to-BamHIMDV DNA) (Fig. 4, lane 9). CEF cells cotransfected with 281MI/1 DNA and pNOTA5, p281MI-1, or pCK300 (Fig. 2) representing the remainder of MDV replicon DNA were not

DpnI

resistant, indicating that they do not contain an origin of replication (Fig. 4, lanes 6 to 8). In all cases, a fraction of input plasmid DNA was susceptible to

DpnI

cleavage and migrated as low-molecular-weight

material,

evident after prolonged exposures of the autoradiogram (data not shown).

Results of

DpnI

resistance assays indicated that a func-tional origin of MDV replication is located in a 700-bp PstI-to-KpnI subfragment of the MDV replicon. The com-plete nucleotide sequence of the 700-bp fragment is shown in Fig. 5. Comparison of pA700 nucleotide sequences with the GenBank data base revealed no significant sequence homol-ogy to any known viral sequences. The exact location of the MDV replication origin in the 281MI/1 viral genome is unknown because of the lack of complete restriction endo-nuclease maps or genomic clones of serotype 2 MDV.

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4.0-..

2.- s

I

_,~~~~~~~~~~~.

1 2 3 4 5 6 7 8 9

FIG. 4. Replicationanalysis of pA5 subclones. Each of the pA5 subclones was tested for replication activity based on the DpnI resistance assay as described in Fig. 3. Each lane contains total transfectedcellular DNA digested withEcoRIandDpnI.32P-labeled pA5 was used to detect plasmid DNA as well as 281MI/1 DNA. Lanes: 1, pA5 Dam'; 2, mock-infected CEF DNA; 3, DNA from CEF cells transfectedwith pA5; 4, DNA from CEF cells transfected with 281MI/1 DNA; 5, DNA from CEF cells cotransfected with 281MI/1DNAandpA5; 6, DNAfromCEF cells cotransfected with 281MI/1DNAand pNOTA5; 7, DNA from CEF cells cotransfected with 281MI/1DNA andp281MI-1; 8, DNA from CEF cells cotrans-fected with 281MI/1 DNA and pCK300; 9, DNA from CEF cells cotransfected with281MI/1 DNAandpA700.

Studies from our laboratory indicate that there are two copies of the replicon sequences within the 281MI/1 viral genome (27). This suggests that there are at least two replication origins in MDV.

HSVcontains two copiesof

oris

within

TRs

and

IRS

and was localized by utilizing HSV amplicons (11, 12). By analogy with HSV, the MDV replicon sequence is most likelytobe present in the repeatsflankingthe

Us

segmentof the MDV genome,assumingthat the structure of serotype 2 MDV DNAis similartothatof serotype 1 and 3MDVDNA. However, on the basis ofDNA sequence identity to lytic origins of alphaherpesvirus replication, Bradleyetal.(3)and Morgan et al. (21) have located aputative serotype 1MDV originofreplicationin repeatsflankingtheUL region.MDV

serotype2functionalreplication originsequences, identified in this study, share 82% identity to theputative serotype 1

MDVreplication

origin

sequences.

In conclusion, we identified a functional origin of MDV replication within a 700-bp subfragment of the MDV repli-con.Systematic deletion

analysis

within the700-bp

fragment

willbe useful todeterminetheminimumsequences

required

for MDV DNAreplication.

Nucleotide sequenceaccession number. The GenBank ac-cession number for the nucleotide sequences reported here

is M74523.

PSTI 20 40 60

CIGCAGVICG GGAAGAGCGG GGGAGGCGCCGICCGAGCG CCGIGAG GTIIGGGG

80 100 120

Cg@3lArG ¶GGIGGGGAAGGGAAGGGG AGACGGCCGA AACCTACGCG T7 CGCGC

140 160 180

TCCGATCCGG GA.TCr-CC GACGGGCt'r C!I' TCCGIATCCG 7CIr'TCCGCCA

200 220 240

CCI'I~CGTC G'IrGCGGGAACCGGACCG GCGCICrAAG CGGAGATCCGGCGC7CCGCr

260 280 300

I I'rA5GACC GGGCCGGI= GAGGGCGTAACGATCAGIG A7GCAATGCAAACGAGCGGG

320 340 360

GCAGACGCGr CAGC_GIIGC

ACCGC:A:ATAAGAT

TATATATATAATATAITAIT

380 400 420

GGCGCAAGGT GCGAACGCCG C CGGGAAGCGG GATCCTAIGC CACLGII1

440 460 480

UGICCGGCCG CGCCCGCGC CGGGCTAGA AAC':3 CCCWC G GGGGCGATC

500 520 540

GGGGACA GGCCrACAAA TACGCGAGCG GPG7CCGGC GGGGACCGT CGIGCIG

560 580 600

GCCGGCCCGC C)G2CCGAAAG CGCGGGACCG CGGTAATAA GCGCCCGCCG TCGCGGATCG

620 640 660

GAi?wlwlIwl3(; lCGTrrACCGCGGGC GAACCGCG GCGAACGAAC CCGITCCCGr

680 700 720

GGGATGCAG GCGGCCGGGA PGCATCGCG CGCCGxTCCC AACGAtG CrACGGCrCG

740 760

C21ItGCGGG GGTCGCCGAC GGGI3GAAOGGGGAP.G1XAC

KNI

FIG. 5. Nucleotide sequences of pA700. pA700, containing a

700-bpsubfragment ofthe MDV replicon DNA,was sequencedin bothorientationsby using dideoxy chain terminations (26). Under-lines represent an MDV replication originsequence that shares a

significantsequenceidentitytoalphaherpesvirus replicationorigins.

Blackbarsindicatethe9-bp motif(CGTTCGCAC) highly conserved among alphaherpesvirus replication origins. Rectangles represent possible Spl-binding sites (GGGCGG), and circles (<>) depict

possible CAAT bindingregions.

We thank A. Finkelstein for providing the pNOTA5 construct.

WealsothankL. F.Velicer andJ. D.Reilly for criticalreviewofthe manuscript.

This research was supported in part by competitive research grants85-CRCR-1-1709 and88-37266-3983 awarded to R. F. Silva andP. M. Coussens,respectively, underthe competitive research grantsadministeredby the U.S. Department ofAgricultureand the MichiganAgriculturalExperimentStation.H.S.Campissupported in part by a Michigan State University biotechnology research fellowshipaward.

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